Substantial physiological information indicates that muscarinic regulation of cardiac (particularly ventricular) function is mediated via muscarinic regulation of the physiological and metabolic effects of catecholamines. A major component of the muscarinic antagonism of beta-adrenergic effects appears to be mediated by attenuation of the catecholamine-induced generation of cyclic AMP. Preliminary data suggest that muscarinic agonists attenuate cyclic AMP generation by inhibiting the activity of adenylate cyclase. The overall objective of this proposal is to elucidate the mechanism by which muscarinic agonists regulate, in an inhibitory manner, cardiac adenylate cyclase activity . The specific aims are: 1) to characterize further the relationship between agonist occupancy of muscarinic receptors and muscarinic inhibition of GTP-activation of adenylate cyclase activity, 2) to elucidate the biochemical mechanisms by which muscarinic agonists modify the ability of GTP to activate adenylate cyclase and decrease beta-adrenergic receptor affinity for catecholamines, and 3) to determine if muscarinic and beta-adrenergic receptors interact with the same regulatory protein (N). The proposed experiments will primarily utilize canine ventricular my ocardial membranes to examine the effects of muscarinic agonists at all known steps in the regulatory GTPase cycle described for the action of beta-adrenergic agonists. The steps to be studied are: 1) quanine nucleotide binding to N, 2) quanine nucleotide dissociation from N, 3) hydrolysis of GTP to GDP at N (GTPase), and 4) guanine nucleotide regulation of the protein-protein interactions between N and beta-adrenergic and muscarinic receptors. The studies will yield further information regarding the basic mechanisms of autonomic regulation of cardiac function and will provide additional bases for understanding of ardiovascular pharmacology. The findings will also be relevant to the general understanding of inhibitory regulation of the enzyme adenylate cyclase.